U.S. patent application number 17/479395 was filed with the patent office on 2022-01-06 for formulation of acoustically activatable particles having low vaporization energy and methods for using same.
The applicant listed for this patent is The Arizona Board of Regents on behalf of the University of Arizona, The Regents of the University of Colorado, a body corporate, The University of North Carolina at Chapel Hill. Invention is credited to Mark A. Borden, Paul A. Dayton, Terry O. Matsunaga, Paul S. Sheeran.
Application Number | 20220000790 17/479395 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-06 |
United States Patent
Application |
20220000790 |
Kind Code |
A1 |
Dayton; Paul A. ; et
al. |
January 6, 2022 |
FORMULATION OF ACOUSTICALLY ACTIVATABLE PARTICLES HAVING LOW
VAPORIZATION ENERGY AND METHODS FOR USING SAME
Abstract
Acoustically activatable particles having low vaporization
energy and methods for making and using same are disclosed. A
particle of material includes a first substance that includes at
least one component that is a gas 25.degree. C. and atmospheric
pressure. A second substance, different from the first substance,
encapsulates the first substance to create a droplet or emulsion
that is stable at room temperature and atmospheric pressure. At
least some of the first substance exists in a gaseous phase at the
time of encapsulation of the first substance within the second
substance to form a bubble. After formation of the bubble, the
bubble is condensed into a liquid phase, which causes the bubble to
transform into the droplet or emulsion having a core consisting of
a liquid. The droplet or emulsion is an activatable phase change
agent that remains a droplet having a core consisting of a liquid
at 25.degree. C. and atmospheric pressure. The first substance has
a boiling point below 25.degree. C. at atmospheric pressure.
Inventors: |
Dayton; Paul A.; (Carrboro,
NC) ; Sheeran; Paul S.; (Durham, NC) ;
Matsunaga; Terry O.; (Tucson, AZ) ; Borden; Mark
A.; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill
The Arizona Board of Regents on behalf of the University of
Arizona
The Regents of the University of Colorado, a body
corporate |
Chapel Hill
Tucson
Denver |
NC
AZ
CO |
US
US
US |
|
|
Appl. No.: |
17/479395 |
Filed: |
September 20, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15247840 |
Aug 25, 2016 |
11123302 |
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17479395 |
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13876165 |
Aug 30, 2013 |
9427410 |
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PCT/US2011/055713 |
Oct 11, 2011 |
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15247840 |
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61505915 |
Jul 8, 2011 |
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61391569 |
Oct 8, 2010 |
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International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 41/00 20060101 A61K041/00; A61K 49/22 20060101
A61K049/22; B01J 13/02 20060101 B01J013/02; A61K 49/00 20060101
A61K049/00; A61K 47/06 20060101 A61K047/06 |
Goverment Interests
GOVERNMENT INTEREST
[0003] This invention was made with government support under Grant
No. EB011704 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for medical diagnostic imaging using activatable
droplets as contrast agents, the method including: producing
encapsulated droplets, each encapsulated droplet comprising a
liquid encapsulated in a shell, wherein the liquid comprises a
liquid phase of a fluorocarbon, a perfluorocarbon, a
chlorofluorocarbon, a hydrofluorocarbon, a hydrocarbon, a gas
having a boiling point that is below room temperature (25.degree.
C.), or combinations thereof; introducing the encapsulated droplets
into a tissue to be imaged; providing activation energy sufficient
to cause the liquid within the encapsulated droplets to change from
a liquid phase to a gas phase, causing the encapsulated droplets to
increase in size and become bubbles of encapsulated gas; and
performing ultrasonic imaging of the tissue using the bubbles as a
contrast agent.
2. A method for medical therapy using activatable droplets, the
method including: producing encapsulated droplets, each
encapsulated droplet comprising a liquid encapsulated in a shell,
wherein the liquid comprises a liquid phase of a fluorocarbon, a
perfluorocarbon, a chlorofluorocarbon, a hydrofluorocarbon, a
hydrocarbon, a gas having a boiling point that is below room
temperature (25.degree. C.), or combinations thereof; delivering
the encapsulated droplets to a target region; and providing
activation energy sufficient to cause the liquid within the
encapsulated droplets to change from a liquid phase to a gas phase,
causing the encapsulated droplets to increase in size and become
bubbles of encapsulated gas, wherein the bubbles obstruct the flow
of blood, oxygen, or nutrients to cells within the target
region.
3. The method of claim 2 wherein the target region includes a
tumor, cancerous cells, or pre-cancerous cells.
4. A method of size selection of particles, comprising: exposing
droplets or emulsions having at least one component that is a gas
at room temperature and atmospheric pressure encapsulated in liquid
form inside a lipid, protein, or polymer capsule particles to at
least one of a pressure other than atmospheric pressure and a
temperature other than room temperature, thereby causing some
portion of the particle distribution to become activated; and
separating the activated particles from the non-activated
particles.
Description
PRIORITY CLAIM
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/247,840, filed Aug. 25, 2016, which is a divisional of
U.S. patent application Ser. No. 13/876,165, filed Mar. 26, 2013,
which is a national stage application under 35 U.S.C. .sctn. 371 of
PCT Patent Application No. PCT/US2011/055713 filed Oct. 11, 2011,
which claims the benefit of
[0002] U.S. Provisional Patent Application Ser. No. 61/505,915
filed, Jul. 8, 2011 and which claims the benefit of U.S.
Provisional Patent Application Ser. No. 61/391,569, filed Oct. 8,
2010; and the disclosures of which are incorporated herein by
reference in their entireties.
GLOSSARY OF TERMS
[0004] The following is a glossary of abbreviations used herein:
[0005] ADV acoustic droplet vaporization [0006] DDFP
dodecafluoropentane (also known as PFP) [0007] DPPC
dipalmitoylphosphatidylcholine
[0008] DPPE-PEG
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] [0009] DFB decafluorobutane (also known as PFB)
[0010] DSPC distearoyl phosphocholine [0011] EPR enhanced
permeability and retention [0012] FDA United States Food and Drug
Administration [0013] HEPES
(4-2-hydroxyethyl)piperazine-1-ethanesulfonic acid [0014] LPC
palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine [0015] MCA
micro-bubble contrast agents [0016] OFP octafluoropropane [0017]
PBS phosphate-buffered saline [0018] PCA phase-change agent [0019]
PCCA phase-change contrast agent [0020] PEG polyethylene glycol
[0021] PFC perfluorocarbon/perfluorochemical--(includes PFB, PFP,
PFMP, and OFP) [0022] PFB perfluorobutane (also known as DFB)
[0023] PFH perfluorohexane [0024] PFMP
perfluoro(2-methyl-3-pentanone) [0025] PFP perfluoropentane (also
known as DDFP) [0026] TAPS trimethylamino propane
BACKGROUND
[0027] The term "bubble" as used herein refers to a bubble of gas
encased or surrounded by an enclosing substance. Bubbles that are
from one micrometer to several tens or hundreds of micrometers in
size are commonly referred to as "microbubbles", while bubbles that
are smaller than one micrometer in size are commonly referred to as
"nanobubbles." The term "droplet" as used herein refers to an
amount of liquid that is encased or surrounded by a different,
enclosing substance. Droplets that are less than one micrometer in
size are commonly referred to as "nanodroplets" and those that are
in the one micrometer to tens or hundreds of micrometers in size
are commonly referred to as "microdroplets." If a droplet is
encased in another liquid, the droplet and its casing may also be
referred to as an "emulsion". The term "particle" as used herein
refers to either a droplet or a bubble of any size.
[0028] Microbubbles for diagnostic ultrasound imaging have been
established in the clinical arena as a sensitive and inexpensive
imaging technique for interrogating landmarks in the vasculature.
Currently, microbubble-enhanced diagnostic ultrasound has been
approved by the FDA for the study of wall motion abnormalities and
ventricular contraction in echocardiography. Researchers have
proposed microbubble-aided ultrasound for a wide range of potential
applications, including functional tumor, kidney, and liver
imaging, identification of vascular inflammation, identification of
vulnerable plaque deposition, thrombus detection and targeted
molecular imaging of angiogenesis. Microbubbles have been used for
therapeutic interventions, primarily in concert with
ultrasound-mediated cavitation for sonothrombolysis.
[0029] Despite their utility as vascular contrast agents and
potential for therapeutic applications, microbubble size (typically
1-5 microns in diameter) prevents their transport outside of the
vasculature, a process commonly referred to as extravasation. In
other words, microbubbles are trapped within the circulatory
system. In order to extravasate into the interstitial space in a
solid tumor, the bubble would need to be smaller than a micron,
i.e., a nanoparticle is required. The exact size limit for
nanoparticle extravasation into the interstitial space in solid
tumors depends on a variety of factors, but has been reported to
fall within the range of 100 nm-750 nm. Nanoparticles make poor
ultrasound contrast agents, however.
[0030] Nanobubbles small enough to diffuse past inter-endothelial
gap junctions scatter ultrasound energy poorly compared to
microbubbles and thus provide limited imaging contrast.
Additionally, bubble circulation in vivo is shown to be on the
order of tens of minutes before bubble dissolution, and clearance
significantly limits contrast enhancement. This short time period
may be insufficient for enough bubbles to accumulate by diffusion
into the tumor interstitium. Droplets of any size provide poor
contrast for ultrasound imaging as compared to equivalently sized
bubbles, and nanodroplets small enough to extravasate into the
interstitial space in solid tumors provide poorer contrast
still.
[0031] One approach to solve the problem of providing ultrasound
contrast agents that are both small enough to extravasate and large
enough to provide sufficient ultrasound contrast has been to
produce a droplet that is small enough to extravasate but which can
be caused to expand into a bubble, a processed referred to as
"activation". Such particles are commonly referred to as "phase
change agents". One method of activation is known as acoustic
droplet vaporization, or ADV. In ADV, the droplet is subjected to
ultrasonic energy, which causes the liquid within the droplet to
change phase and become a gas. This causes the droplet to become a
bubble, with the corresponding increase in size. The ultrasound
impulses impart a mechanical pressure upon the tissues, and the
amount of pressure applied is indicated in terms of a mechanical
index, or MI.
[0032] Particles that start as droplets but can be activated to
become bubbles are referred to as "metastable", because they are
stable as droplets (e.g., they don't spontaneously expand into
bubbles) without additional energy. If these PCAs are used as
contrast agents, they are commonly referred to as "phase-change
contrast agents" (PCCAs).
[0033] Recently there has been interest in the use of PFC droplets
for this purpose. To date, PCAs have been developed using PFCs
which have boiling points above room temperature (25.degree. C.),
which are herein referred to as "low volatility PFCs". Examples
include dodecafluoropentane (DDFP), perfluorohexane (PFH), and
perfluoroheptane. These low volatility PRCs have been used to make
PCAs that have a diameter greater than 1 micron, i.e.,
microdroplets which activate into microbubbles. PFCs with boiling
points below room temperature, which are herein referred to as
"high volatility" or "highly volatile" PFCs, have not been used to
make microparticles out of a concern that, if subjected to body
temperature (37.degree. C.), a droplet containing a highly volatile
PFC might spontaneously change phase.
[0034] However, the low-volatility PFCs conventionally used to make
micro-PCAs are not suitable for making nano-PCAs. Many in vitro
studies have shown that the energy required to activate a PFC-based
PCA increases as the diameter of the initial droplet decreases.
There is a direct correlation between activation energy and
mechanical index, and applications involving relatively low
frequencies and/or sub-micron droplets may require pressures higher
than diagnostic ultrasound machines currently provide. This is an
obstacle to human treatment, because excessive ultrasonic
activation energy can cause tissue damage or other unwanted
bioeffects.
[0035] Thus, PFCs that had been used in microbubbles may be
unsuitable for use in nanodroplets due to the excessive activation
energy required. The smaller the nanodroplet, the more activation
energy is required, and the less suitable the PFC. For example, the
Antoine vapor pressure equation was analyzed in order to assess the
theoretical vaporization temperature dependence upon droplet
diameter of selected PFCs as a result of the influence of
interfacial surface tension. Using this model to investigate the
influence of PFC boiling points, it was concluded that DDFP, PFH,
and perfluoroheptane may require a relatively large amount of
energy in order to elicit droplet vaporization at a size that would
practically be able to extravasate through endothelial gap
junctions and into the extravascular space.
[0036] Therefore, there exists a need for a phase-change agent that
is stable at physiological temperatures yet is more susceptible to
ultrasound pressures. Such a particle could provide a more
efficacious vehicle for extravasation into tissue and activation at
the site of action in many applications. For human therapeutic and
diagnostic use, there is a need for a stable nanoparticle capable
of being vaporized using frequencies and mechanical indices within
the FDA-approved limits of commercial clinical diagnostic
ultrasound machines.
SUMMARY
[0037] The subject matter described herein includes formulation
methods and applications for particles that can be activated by
acoustic energy to convert from a liquid state to a gas state. In
one embodiment, nanoparticles suitable for use in human
diagnostics, imaging, therapeutics, and treatment are presented.
The methods described herein produce stabilized nano- and
micro-particles, in liquid or emulsion form, of compounds that are
normally gas at room temperature and atmospheric pressure. Two
distinct methods are disclosed: the first is called the "droplet
extrusion" method and the second is called the "bubble
condensation" method.
[0038] In one embodiment, the droplet extrusion method includes
causing the first substance to condense into a liquid and then
extruding or emulsifying the first substance into or in the
presence of a second substance to create droplets or emulsions in
which the first substance is encapsulated by the second substance.
To condense the first substance, it may be cooled to a temperature
below the phase transition temperature of the component having the
lowest boiling point, it may be compressed to a pressure above the
phase transition pressure of the component having the highest phase
transition pressure value, or a combination of the above. The
contents of the droplet or emulsion so formed may be entirely or
primarily in a liquid phase.
[0039] In one embodiment, the bubble condensation method includes
extruding or emulsifying the first substance into or in the
presence of the second substance to create bubbles having an outer
shell of the second substance encapsulating an amount of the first
substance, at least some of which is in gaseous form. The bubble
thus formed is cooled and/or compressed such that the contents of
the bubble reach a temperature below the phase transition
temperature of the component having the lowest boiling point at
that pressure. This causes the gas within the bubble to condense to
a liquid phase, which transforms the bubble into a droplet or
emulsion. In this manner, droplets or emulsions in which the first
substance is encapsulated by the second substance are created.
[0040] The two methods described above produce particles containing
in liquid form a substance that is normally a gas at room
temperature and pressure, and stabilizing these particles in their
liquid form using a shell such as a lipid, protein, polymer, gel,
surfactant, or sugar. The surface tension of the shell enables
these particles to be stable in liquid form, even when the
surrounding temperature is raised back to room temperature.
Acoustic energy can then "activate" the particle, returning it to
gas form.
[0041] In one embodiment, a method for delivery of particles to a
target region includes introducing particles comprising stable,
activatable nanodroplets, each nanodroplet comprising a liquid
encapsulated in a shell, where the liquid comprises at least one
component that is a gas at room temperature and atmospheric
pressure, into a blood vessel in the vicinity of a target region.
The particles then extravasate into the target region.
[0042] In one embodiment, a method for medical diagnostic imaging
using activatable droplets as contrast agents includes producing
encapsulated droplets, each encapsulated droplet containing a
liquid encapsulated in a shell, where the liquid includes a liquid
phase of a fluorocarbon, a perfluorocarbon, a chlorofluorocarbon, a
hydrofluorocarbon, a hydrocarbon, a gas having a boiling point that
is below room temperature (25.degree. C.), or combinations thereof;
introducing the encapsulated droplets into a tissue to be imaged;
providing activation energy sufficient to cause the liquid within
the encapsulated droplets to change from a liquid phase to a gas
phase, causing the encapsulated droplets to increase in size and
become bubbles of encapsulated gas; and performing ultrasonic
imaging of the tissue using the bubbles as a contrast agent.
[0043] In one embodiment, a method for medical therapy using
activatable droplets includes producing encapsulated droplets, each
encapsulated droplet including a liquid encapsulated in a shell,
where the liquid comprises a liquid phase of a fluorocarbon, a
perfluorocarbon, a chlorofluorocarbon, a hydrofluorocarbon, a
hydrocarbon, a gas having a boiling point that is below room
temperature (25.degree. C.), or combinations thereof; including a
therapeutic agent in or on the shell; delivering the encapsulated
droplets to a target region; and providing activation energy
sufficient to cause the liquid within the encapsulated droplets to
change from a liquid phase to a gas phase, causing the encapsulated
droplets to increase in size and become bubbles of encapsulated
gas, wherein the substance to be delivered to the target tissue
enters into the cells of the target tissue.
[0044] In one embodiment, a method for medical therapy using
activatable droplets includes producing encapsulated droplets, each
encapsulated droplet including a liquid encapsulated in a shell,
where the liquid includes a liquid phase of a fluorocarbon, a
perfluorocarbon, a chlorofluorocarbon, a hydrofluorocarbon, a
hydrocarbon, a gas having a boiling point that is below room
temperature (25.degree. C.), or combinations thereof; delivering
the encapsulated droplets to a target tissue; and providing
activation energy sufficient to cause the liquid within the
encapsulated droplets to change from a liquid phase to a gas phase,
causing the encapsulated droplets to increase in size and become
bubbles of encapsulated gas, where the bubbles obstruct the flow of
blood, oxygen, or nutrients to a target region.
[0045] In one embodiment, a method of size selection of particles
including droplets or emulsions having at least one component that
is a gas at room temperature and atmospheric pressure encapsulated
in liquid form inside a lipid, protein, or polymer capsule,
includes exposing the particles to at least one of a pressure other
than atmospheric pressure and a temperature other than room
temperature, thereby causing some portion of the particle
distribution to become activated, and separating the activated
particles from the non-activated particles.
[0046] Acoustically activatable particles having low vaporization
energy and methods for making and using same are disclosed. A
particle of material includes a first substance that includes at
least one component that is a gas 25.degree. C. and atmospheric
pressure. A second substance, different from the first substance,
encapsulates the first substance to create a droplet or emulsion
that is stable at room temperature and atmospheric pressure. At
least some of the first substance exists in a gaseous phase at the
time of encapsulation of the first substance within the second
substance to form a bubble. After formation of the bubble, the
bubble is condensed into a liquid phase, which causes the bubble to
transform into the droplet or emulsion having a core consisting of
a liquid. The droplet or emulsion is an activatable phase change
agent that remains a droplet having a core consisting of a liquid
at 25.degree. C. and atmospheric pressure. The first substance has
a boiling point below 25.degree. C. at atmospheric pressure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] Preferred embodiments of the subject matter described herein
will now be explained with reference to the accompanying drawings,
wherein like reference numerals represent like parts, of which:
[0048] FIG. 1 is a graph illustrating the relationship between
droplet diameter, shown on the X-axis, and predicted vaporization
temperature, shown on the Y-axis, for lipid-encapsulated
nanodroplets, each containing one of four common PFCs;
[0049] FIG. 2 is a flow chart illustrating an exemplary process for
preparing particles of materials having a first substance that is
enclosed by second substance that acts as an encapsulating
material, where the first substance includes at least one component
that is a gas at room temperature and atmospheric pressure,
according to an embodiment of the subject matter described
herein;
[0050] FIG. 3 is a flow chart illustrating an exemplary process for
preparing particles of materials having a first substance that is
enclosed by second substance that acts as an encapsulating
material, where the first substance includes at least one component
that is a gas at room temperature and atmospheric pressure,
according to another embodiment of the subject matter described
herein;
[0051] FIG. 4 is a graph showing an observed relationship between
initial diameter of a droplet generated according to methods
described herein and the mechanical index required to vaporize
it;
[0052] FIG. 5 is a graph illustrating activation energy for
droplets containing DFB, droplets containing OFP, and droplets
containing a 50%/50% mix of DFB and OFP, generated using the bubble
condensation method according to an embodiment of the subject
matter described herein;
[0053] FIG. 6 is a flow chart illustrating an exemplary process for
delivery of particles to a target region according to an embodiment
of the subject matter described herein;
[0054] FIG. 7 is a flow chart illustrating an exemplary process for
medical diagnostic imaging using activatable droplets as contrast
agents according to an embodiment of the subject matter described
herein;
[0055] FIG. 8 is a flow chart illustrating an exemplary process for
medical therapy using activatable droplets as a vehicle for
delivering therapeutic agents according to an embodiment described
herein ;
[0056] FIG. 9 is a flow chart illustrating an exemplary process for
medical therapy using activatable droplets to obstruct the flow of
blood, oxygen, or nutrients to cells, such as tumor tissues,
according to an embodiment of the subject matter described herein;
and
[0057] FIG. 10 is a flow chart illustrating an exemplary process
for size selection of particles according to an embodiment
described herein.
DETAILED DESCRIPTION
[0058] An ideal phase change agent for use where extravasation into
interstitial regions of tissue is desired, such as an extravascular
ultrasound contrast agent, for applications where thermal and
cavitation-based bioeffects are minimized should be: 1) stable in
the vasculature for a sufficient time period, 2) capable of
extravasation out of the vascular space, and: 3) labile enough to
be activated and interrogated by clinical ultrasound machines at
clinically relevant acoustic intensities. The subject matter
described herein includes methods to produce acoustically
activatable nanoparticles that are formulated with high volatility
PFCs yet remain stable at room temperature and pressure. The
resulting droplets are acoustically activatable with substantially
less energy than other favored compounds proposed for phase-change
contrast agents. Also presented are uses of these nanoparticles in
medical diagnostics, imaging, therapy, and treatment. In one
embodiment, the activation energy of the nanoparticle may be tuned
to a particular value, opening up the possibility of
highly-targeted treatments.
[0059] To determine which PFCs had potential as a phase-change
contrast agent at physiological temperatures and with
pre-activation droplet size in the range desired for extravasation,
it was necessary to estimate the energy that would be required to
activate droplets containing the PFCs. The energy required depends
on both the PFC contained within the droplet and the diameter of
the droplet itself. To estimate this energy, calculations were
performed using the Antoine vapor-pressure equation. This equation
was derived from the Clausius-Clapeyron relation by Antoine in
1888, and when re-arranged for temperature is expressed as:
T = B A - log 1 .times. 0 .times. P - C ( Eq . .times. #1 )
##EQU00001##
where P is pressure, T is temperature, and A, B, and C are
gas-dependent constants observed to be valid for a particular
temperature range. This equation uses experimental results to
develop a basic relationship between temperature and pressure as a
droplet of a particular substance vaporizes. A droplet will
experience an additional pressure due to interfacial surface
tension effects, defined as the Laplace pressure:
.DELTA. .times. .times. P = P inside - P outside = 2 .times.
.sigma. r ( Eq . .times. #2 ) ##EQU00002##
where r is the radius of the droplet, .sigma. is surface tension,
and P.sub.inside and P.sub.outside represent the pressure inside
the droplet core and the ambient pressure in the surrounding media,
respectively. PFCs typically have fairly low surface tension values
on the order of 10 mN/m at room temperature.
[0060] Because the Laplace pressure is an inverse function of
radius, smaller droplets will experience greater pressure.
Encapsulating the droplets in a lipid or polymer shell stabilizes
the droplets from coalescence and alters the interfacial surface
tension. Depending on the properties of the encapsulating shell, a
larger resulting surface tension may cause an increase in the
pressure exerted, which essentially increases the vaporization
temperature of the droplet.
[0061] In designing agents for human medical imaging purposes, the
ambient pressure may be defined as:
P.sub.amb=P.sub.atm+P.sub.body (Eq. #3)
where P.sub.atm=101.325 kPa and P.sub.body is a representative
pressure inside the human body (vascular or other). Although
intravascular pressure is inherently pulsatile, for the purposes of
these calculations, an average value of P.sub.body=12.67 kPa was
used. With a total pressure exerted on the droplet of:
P = P amb + .DELTA. .times. P = P atm + P body + 2 .times. .sigma.
r ( Eq . .times. #4 ) ##EQU00003##
the resulting modified Antoine vapor-pressure equation is:
T = B A - log 1 .times. 0 ( P atm + P b .times. o .times. d .times.
y + 2 .times. .sigma. r ) - C ( Eq . .times. #5 ) ##EQU00004##
[0062] Published surface tensions often vary between 25 mN/m and
50-60 mN/m, depending on surfactant properties. Although the exact
surface tension of lipid solutions used in published studies was
not known, a value near 51 mN/m was sufficient for the purposes of
these initial calculations in that it provided a Laplace pressure
near the upper limit of what can be expected. The constants A,B,C
were gathered from the National Institute of Standards and
Technology (NIST) Chemistry WebBook (Linstrom and Mallard 2010) for
the nearest available temperature range. The results of these
calculations is shown in FIG. 1.
[0063] FIG. 1 is a graph illustrating the relationship between
droplet diameter, shown on the X-axis, and predicted vaporization
temperature, shown on the Y-axis, for lipid-encapsulated
nanodroplets, each containing one of four common PFCs:
perfluorohexane (PFH), dodecafluoropentane (DDFP), decafluorobutane
(DFB), and octafluoropropane (OFP), shown with human body
temperature for comparison. The natural boiling points of PFH,
DDFP, DFB, and OFP are 56.6.degree. C., 29.degree. C., -1.7.degree.
, and -37.6.degree. C., respectively.
[0064] In order to estimate the size of the bubble produced by
activating a droplet, ideal gas laws (PV=nRT, where n, P, V, and T
represent the number of moles of PFC, pressure, volume, and
temperature, respectively) can be used to approximate the expansion
factor when a liquid undergoes a phase conversion to the gaseous
state. Because perfluorocarbons are immiscible in the liquid state
and have low diffusivity in the gaseous state, here it is assumed
that the number of moles is constant from the liquid phase to the
gaseous phase (n.sub.l=n.sub.g). The moles of PFC in the spherical
droplet can be computed as:
n 1 = 4 .times. .pi. .times. r 1 3 .times. .rho. 1 3 .times. M ( Eq
. .times. #6 ) ##EQU00005##
where r.sub.l is the radius of the liquid droplet, .rho..sub.l is
the liquid density, and M is the molar mass. Substituting this into
the ideal gas law and simplifying as a ratio of the gas-phase
radius to liquid-phase radius gives:
r g r 1 = .rho. 1 .times. R .times. T M .times. P 3 ( Eq . .times.
#7 ) ##EQU00006##
[0065] Expanding with Eq. #4 gives
r g r 1 = .rho. 1 .times. RT M ( P a .times. t .times. m + P b
.times. o .times. d .times. y + 2 .times. .sigma. r g ) 3 ( Eq .
.times. #8 ) ##EQU00007##
As r.sub.g approaches very large values, the surface tension
component becomes negligible.
[0066] Decafluorobutane has a molar mass of M=0.238 kg/mol, and at
37.degree. C. (310 K) .rho..sub.l.apprxeq.1500 kg/m.sup.3.
Evaluating Eq. #8 with in vivo (P.sub.body=12.67 kPa) and in vitro
(P.sub.body=0 kPa) conditions and neglecting surface tension
effects reveals that, based on the assumptions given, a droplet of
DFB can be predicted to expand to an approximate upper limit of 5.2
to 5.4 times its original diameter once vaporized (neglecting any
deviations from ideal gas laws). Rearranging Eq. #8 such that it is
solved for liquid droplet radius becomes:
r 1 = M .times. r g 2 .function. [ r g .function. ( P a .times. t
.times. m + P b .times. o .times. d .times. y ) + 2 .times. .sigma.
] .rho. 1 .times. RT 3 ( Eq . .times. #9 ) ##EQU00008##
[0067] This allows one, based on ideal gas laws and surface tension
effects, to estimate the size of the droplet that vaporized to
become a bubble of a known size. Eq. #8 can also be solved for
r.sub.g, providing a numerically equivalent--though much more
complex--solution. For the purposes of this study, Eq. #9 becomes a
more convenient solution, as measured bubble sizes are used to
estimate originating droplet sizes.
[0068] While the constants used are not expected to predict the
vaporization relationship completely accurately in the desired
temperature range, the calculation shows DFB droplets appear to
have the potential to remain stable in the 200-600 nm diameter
range at temperatures just above body temperature and that the
bubble created by activating the droplet will be of sufficient size
to effectively perform as an ultrasound contrast agent. The
calculation also shows that OFP droplets have the potential to
remain stable at sizes below 200 nm, although the -37.6.degree. C.
boiling point presents significant generation challenges. However,
no method to create DFB or OFP droplets in the sub-micron size
range was known or found in the prior art.
[0069] The subject matter described herein includes two methods
creating stable droplets or emulsions, including particles less
than 1 micron in diameter, that contain PFCs with low boiling
points, including PFCs with boiling points that are below body
temperature (37.degree. C.) or below room temperature (25.degree.
C.), by encapsulating the particles in a lipid, protein, polymer,
gel, surfactant, peptide, or sugar. It has been shown that this
technique may be used to successfully create stable nanodroplets
containing DFP, OFP, or a mixture of the two, with and without
other substances, where the substance being encapsulated would
otherwise vaporize without the stabilizing encapsulation. The
nanodroplets so created have activation energies that are low
enough that the nanodroplets may be used for human diagnostics,
therapeutics, and treatment. The subject matter described herein
also includes methods of using these nanodroplets for diagnostics,
therapeutics, and other treatments.
Droplet Extrusion Method
[0070] FIG. 2 is a flow chart illustrating an exemplary process for
preparing particles of materials having a first substance that is
enclosed by second substance that acts as an encapsulating
material, where the first substance includes at least one component
that is a gas at room temperature and atmospheric pressure,
according to an embodiment of the subject matter described herein.
Example particles include droplets or emulsions. The first
substance may be herein referred to as, for example, "the
encapsulated substance", "the contents", "the filler", "the
filling", "the core", and the like. The second substance may be
herein referred to as, for example, "the encapsulating substance",
"the encapsulation material", "the capsule", "the container", "the
shell", and the like.
[0071] At block 200, the first substance condensed to a liquid
phase. This may be done, for example, by cooling the first
substance to a temperature below the phase transition temperature
of the component having the lowest boiling point, by compressing
the first substance to a pressure that is above the phase
transition pressure of the component having the highest phase
transition pressure, or a combination of the above.
[0072] At block 202, the first substance is extruded into or in the
presence of the second substance to create droplets or emulsions in
which the first substance is encapsulated by the second substance.
In one embodiment, the contents of the droplet or emulsion is
entirely or primarily in a liquid phase.
[0073] In one embodiment the particles are extruded at a
temperature below the phase transition temperature of the component
having the lowest boiling point. In one embodiment, the particles
are formed through a flow-focusing junction in a microfluidic
device, where the device is maintained at a temperature below the
phase transition temperature of the component having the lowest
boiling point.
[0074] In one embodiment, the particles are extruded in a
pressurized environment, where the ambient pressure is above phase
transition pressure of the component having the highest phase
transition pressure. In one embodiment, the particles are extruded
at a temperature that is either above or below the boiling point of
the component having the lowest boiling point. In one embodiment,
the particles are extruded at a temperature that is below the
boiling point of the component having the lowest boiling point.
[0075] In one embodiment, the preparation involves shaking. In one
embodiment, the preparation involves extrusion through a filter. In
one embodiment, the filter has a pore size greater than the size of
the desired particle. In one embodiment, the pore size is 10 times
greater than the desired particle size. In one embodiment, the pore
size is 2.about.7 times greater than the desired particle size. In
one embodiment, the pore size is 3.about.6 times greater than the
desired particle size. In one embodiment, the pore size is 5 times
greater than the desired particle size.
[0076] In one embodiment, the first substance being encapsulated
includes a gas, such as a fluorocarbon, a perfluorocarbon, a
chlorofluorocarbon, a hydrofluorocarbon, a hydrocarbon, a gas
having a boiling point that is below room temperature (25.degree.
C.), or combinations of the above, that is condensed to a liquid
phase and encapsulated. In one embodiment, the encapsulated
droplets are activatable particles, such as phase change agents, or
PCAs. The PCAs may be activated by exposure to ultrasonic, X-ray,
optical, infrared, microwave, or radio frequency energy.
[0077] In one embodiment, the gas has a boiling point temperature
in a range from approximately 50.degree. C. to 40.degree. C. at
atmospheric pressure. In one embodiment, the gas has a boiling
point temperature in a range from approximately 40.degree. C. to
30.degree. C. at atmospheric pressure. In one embodiment, the gas
has a boiling point temperature in a range from approximately
30.degree. C. to 20.degree. C. at atmospheric pressure. In one
embodiment, the gas has a boiling point temperature in a range from
approximately 20.degree. C. to 10.degree. C. at atmospheric
pressure. In one embodiment, the gas has a boiling point
temperature in a range from approximately 10.degree. C. to
0.degree. C. at atmospheric pressure. In one embodiment, the gas
has a boiling point temperature in a range from approximately
0.degree. C. to 10.degree. C. at atmospheric pressure. In one
embodiment, the gas has a boiling point temperature in a range from
approximately 10.degree. C. to 20.degree. C. at atmospheric
pressure.
[0078] In one embodiment, encapsulating droplets of the liquid
phase in the encapsulation material includes extruding or
emulsifying the liquid phase using a microfluidics technique to
produce droplets of the liquid phase and encapsulating the droplets
of the liquid phase in the encapsulation material. In one
embodiment, using a microfluidics technique comprises using a
flow-focusing junction or a T-junction in a microfluidic device. In
one embodiment, the device is maintained at a temperature below the
phase transition temperature of the component having the lowest
boiling point.
[0079] In one embodiment, at least some of the droplets range from
approximately 10 um to 50 um in diameter. In one embodiment, at
least some of the droplets range from approximately 5 um to 10 um
in diameter. In one embodiment, at least some of the droplets range
from approximately 1 um to 5 um in diameter. In one embodiment, at
least some of the droplets range from approximately 800 nm to 1 um
in diameter. In one embodiment, at least some of the droplets range
from approximately 600 nm to 800 nm in diameter. In one embodiment,
at least some of the droplets range from approximately 400 nm to
600 nm in diameter. In one embodiment, at least some of the
droplets range from approximately 200 nm to 400 nm in diameter. In
one embodiment, at least some of the droplets range from
approximately 100 nm to 200 nm in diameter. In one embodiment, at
least some of the droplets range from approximately 50 nm to 100 nm
in diameter.
[0080] In one embodiment, the encapsulation material comprises a
lipid, protein, polymer, gel, surfactant, peptide, or sugar. In one
embodiment, the encapsulation material comprises lung surfactant
proteins or their peptide components to form and stabilize bilayer
and multilayer folds of the encapsulation material attached to
maintain enough encapsulation material sufficient to fully
encapsulate the liquid phase before droplet vaporization and the
gas phase following vaporization. Example surfactants include
amphiphilic polymers and copolymers, amphiphilic peptides,
amphiphilic dendrimers, amphiphilic nucleic acids, and other
amphiphiles.
[0081] In one experiment, this droplet extrusion method produced a
highly varying size distribution of viable droplets, from droplets
near the optical resolution of the test equipment (2.times.3
microns in diameter) to droplets more than 10 microns in diameter.
The droplets so produced were stable at 37.degree. C. and could be
subsequently vaporized by ultrasonic energy
Bubble Condensation Method
[0082] FIG. 3 is a flow chart illustrating an exemplary process for
preparing particles of materials having a first substance that is
enclosed by second substance that acts as an encapsulating
material, where the first substance includes at least one component
that is a gas at room temperature and atmospheric pressure,
according to another embodiment of the subject matter described
herein. At block 300, the first substance extruded into or in the
presence of the second substance to create bubbles having an outer
shell of the second substance encapsulating an amount of the first
substance, at least some of which is in gaseous form. In one
embodiment, the contents of the bubble are entirely or primarily in
a gaseous phase. At block 302, the bubble thus formed is cooled
and/or compressed such that the contents of the bubble reach a
temperature below the phase transition temperature of the component
having the lowest boiling point at that pressure. This causes the
gas within the bubble to condense to a liquid phase, which
transforms the bubble into a droplet or emulsion. In this manner,
droplets or emulsions in which the first substance is encapsulated
by the second substance are created. This method offers the
advantage of making smaller, more uniform droplet sizes with peaks
on the order of 200-300 nm--small enough for potential
extravasation into solid tumors.
[0083] In one embodiment, the first substance includes a gas, such
as a fluorocarbon, a perfluorocarbon, a chlorofluorocarbon, a
hydrofluorocarbon, a hydrocarbon, a gas having a boiling point that
is below room temperature (25.degree. C.), or combinations of the
above. In one embodiment, the encapsulated droplets are activatable
particles, such as phase change agents, or PCAs. The PCAs may be
activated by exposure to ultrasonic, X-ray, optical, infrared,
microwave, or radio frequency energy.
[0084] In one embodiment, the gas has a boiling point temperature
in a range from approximately 50.degree. C. to 40.degree. C. at
atmospheric pressure. In one embodiment, the gas has a boiling
point temperature in a range from approximately 40.degree. C. to
30.degree. C. at atmospheric pressure. In one embodiment, the gas
has a boiling point temperature in a range from approximately
30.degree. C. to 20.degree. C. at atmospheric pressure. In one
embodiment, the gas has a boiling point temperature in a range from
approximately 20.degree. C. to 10.degree. C. at atmospheric
pressure. In one embodiment, the gas has a boiling point
temperature in a range from approximately 10.degree. C. to
0.degree. C. at atmospheric pressure. In one embodiment, the gas
has a boiling point temperature in a range from approximately
0.degree. C. to 10.degree. C. at atmospheric pressure. In one
embodiment, the gas has a boiling point temperature in a range from
approximately 10.degree. C. to 20.degree. C. at atmospheric
pressure.
[0085] In one embodiment, creating bubbles of a gas encapsulated in
an encapsulation material includes extruding or emulsifying the gas
in the presence of lipids. In one embodiment, creating bubbles of a
gas encapsulated in an encapsulation material includes extruding or
emulsifying the gas in a HEPES buffer. In one embodiment, creating
bubbles of a gas encapsulated in an encapsulation material includes
extruding or emulsifying the gas in a buffer having a pH in a range
from approximately 3 to 9. In one embodiment, creating bubbles of a
gas encapsulated in an encapsulation material includes extruding or
emulsifying the gas in a buffer having a pH in a range from
approximately 6 to 8.
[0086] In one embodiment, condensing the encapsulated gas into a
liquid phase includes cooling the bubbles under pressure until the
encapsulated gas condenses into a liquid phase. In one embodiment,
the bubbles are cooled to a temperature in a range from
approximately 0.degree. C. to 10.degree. C. In one embodiment, the
bubbles are cooled to a temperature in a range from approximately
10.degree. C. to 0.degree. C. In one embodiment, the bubbles are
cooled to a temperature in a range from approximately 20.degree. C.
to 10.degree. C. In one embodiment, the bubbles are exposed to a
pressure that is greater than 50 psi. In one embodiment, the
bubbles are exposed to a pressure that is greater than 20 psi. In
one embodiment, the bubbles are exposed to a pressure that is
greater than 10 psi. In one embodiment, the bubbles are exposed to
a pressure that is in a range from approximately 10 psi to 20 psi.
In one embodiment, the bubbles are exposed to a pressure that is in
a range from approximately 20 psi to 50 psi. In one embodiment, the
bubbles are exposed to a pressure that is in a range from
approximately 50 psi to 100 psi. In one embodiment, the bubbles are
exposed to a pressure that is in a range from approximately 100 psi
to 200 psi. In one embodiment, the bubbles are exposed to a
pressure that is in a range from approximately 200 psi to 500
psi.
[0087] In one embodiment, at least some of the bubbles range from
approximately 10 um to 50 um in diameter. In one embodiment, at
least some of the bubbles range from approximately 5 um to 10 um in
diameter. In one embodiment, at least some of the bubbles range
from approximately 1 um to 5 um in diameter. In one embodiment, at
least some of the droplets range from approximately 800 nm to 1 um
in diameter. In one embodiment, at least some of the droplets range
from approximately 600 nm to 800 nm in diameter. In one embodiment,
at least some of the droplets range from approximately 400 nm to
600 nm in diameter. In one embodiment, at least some of the
droplets range from approximately 200 nm to 400 nm in diameter. In
one embodiment, at least some of the droplets range from
approximately 100 nm to 200 nm in diameter. In one embodiment, at
least some of the droplets range from approximately 50 nm to 100 nm
in diameter.
[0088] In one embodiment, the encapsulation material includes a
lipid, protein, polymer, gel, surfactant, peptide, or sugar. In one
embodiment, the encapsulation material includes lung surfactant
proteins or their peptide components to form and stabilize bilayer
and multilayer folds of the encapsulation material attached to
maintain enough encapsulation material sufficient to fully
encapsulate the liquid phase before droplet vaporization and the
gas phase following vaporization.
[0089] For both the droplet extrusion method and the bubble
condensation method, the first substance may include a PFC that has
a phase transition temperature that is below room temperature or
below human body temperature of 37.degree. C. at normal atmospheric
pressure. For example, the first substance may include a highly
volatile compound, such as DFP, OFP, a mixture of the two, or a
mixture of DFP and/or OFP with another PFC. The first substance may
also be a mixture of DFP and/or OFP with third substance, where the
third substance may or may not be a gas at room temperature or body
temperature. In one embodiment, the second substance may be made up
of lipids, proteins, polymers, a gel, a surfactant, a peptide, a
sugar, another suitable encapsulating material, or a combination of
the above.
[0090] Whether the method of FIG. 2 or the method of FIG. 3 is
used, the resulting droplets are stabile at room temperature/body
temperature and pressure. Droplets containing DFB, OFP, or a
combination have an activation energy that is low enough for use in
human diagnostics, therapeutics, or treatment. For example,
droplets containing DFP have the desired low vaporization
threshold, even when prepared as sub-micron droplets. DFB's boiling
point of -1.7.degree. C. is significantly lower than other PFCs
commonly used in ADV, which allows vaporization at much lower
pressures than similarly-sized emulsions of higher boiling-point
PFCs. Lipid-encapsulated nanodroplets containing condensed OFP,
which has a boiling point of -40.degree. C., are surprisingly
stabile: exposing these nanodroplets to body temperature is not by
itself enough to cause them to activate and expand into
microbubbles; additional energy, such as may be provided by a
medical ultrasound transceiver, is required. The resulting droplets
are activatable with substantially less energy than other favored
PCCA compounds. For example, when exposed in vitro to a 2 .mu.s
ultrasound pulse at 5 MHz and MI=1.2, the generated nanodroplets
yield a distribution of microbubbles that corresponds well with
expected expansion of the initial droplets through ideal gas law
predictions with surface tension effects included.
[0091] The methods described in FIGS. 2 and 3 can be used to
produce stabilized, lipid-encapsulated nanodroplets of highly
volatile compounds suitable for use as extravascular ultrasound
contrast agents and activatable using ADV at diagnostic ultrasound
frequencies and mechanical indices within FDA guidelines for
diagnostic imaging. The methods described above have routinely
yielded droplets in the 200.times.300 nm range, which, upon
activation, become bubbles between 1 and 5 microns in size. In
other words, the droplets are small enough to extravasate, and,
once activated, the bubbles are large enough to provide sufficient
contrast for ultrasound imaging. As will be described in more
detail below, the particles thus created are suitable for use in
diagnostics, therapeutics, and treatment other than ultrasound
imaging, such as drug and gene delivery.
[0092] It is noted, however, that the methods and techniques
described herein may be used to create stable nanodroplets
containing highly volatile PFCs, even those that have activation
energies above the limits defined for human medical use. The
unexpected stability of OFP droplets in the sub-micron range
indicate that other highly volatile PFCs may be used to create
nanodroplets that may be used to interrogate materials other than
biological tissues, for example, to which a higher activation
energy may be applied, e.g., where there is no concern about
bioeffects.
Detailed Preparation
[0093] In one embodiment, lipid films were prepared with a lipid
composition containing 85 mole percent DPPC, 10 mole percent LPC,
and 5 mole percent DPPE-PEG 2000. The lipids were dissolved in less
than 1 mL of chloroform and gently evaporated with nitrogen gas.
The lipids were kept under a lyophilzer overnight in order to
remove residual solvent and to create lipid films. The lipid films
were rehydrated with approximately 1 mL of HEPES buffer (pH=7.4)
and sonicated for 10 minutes in a water bath sonicator at
50-60.degree. C. The rehydrated films were subjected to 10
freeze-thaw cycles where the freezing section consisted of an
isopropanol bath with dry ice and the thawing section was a
50-60.degree. C. water bath. This created a homogeneous lipid
suspension, which was also stirred for 10 minutes at 50-60.degree.
C. immediately afterwards. The resulting concentration of the lipid
solution was about 20 mg/m L.
[0094] For the method described in FIG. 2, a PFC, such as DFB, was
condensed in a container over dry ice. The condensed DFB was poured
into a 2 mL glass vial and crimped. Two hundred microliters (200
.mu.l) of DFB was then mixed with the lipid solution and the
samples were extruded in a -20.degree. C. cold room by 20 passes
through a 1 .mu.m porous membrane filter. After extrusion, the
resulting emulsion was stored at 4.degree. C. in a crimped 2 mL
vial. Samples were observed throughout the extrusion process to
make sure they did not freeze.
[0095] For the method described in FIG. 3, microbubbles of a PFC,
such as DFB, were formulated by the dissolution DPPC,
DPPE-PEG-2000, and TAPS in a molar ratio of 65:5:30
(mole:mole:mole) and a total lipid concentration of 0.75 mg/mL, 1.5
mg/mL, and 3 mg/mL. The excipient liquid was comprised of propylene
glycol, glycerol, and normal saline. Microbubbles were formed via
agitation by shaking for 45 seconds. The 2 mL vial containing the
formed microbubbles was then immersed in a CO.sub.2/isopropanol
bath controlled to a temperature of approximately -5.degree. C. A
25 G syringe needle containing 30 mL of room air was then inserted
into the vial septum and the plunger depressed slowly. This step
was repeated with another 30 mL of room air. Lipid freezing was
avoided by observing the contents of the vial as well as the
temperature of the CO.sub.2/isopropanol solution periodically.
After pressurizing with a total of 60 mL of room air, the syringe
needle was removed from the vial, leaving a pressure head on the
solution.
Analysis of Results
[0096] The vaporization threshold of individual droplets was
determined, and the size of the bubble resulting from activation of
the droplet was measured. The vaporization threshold was determined
by observing what level of ultrasound pressure was required to
cause droplets of various diameters to activate. The measured
pressure that induced vaporization was converted into a mechanical
index (MI), defined as:
Peak .times. .times. Negative .times. .times. Pressure .times.
.times. ( MPa ) US .times. .times. Frequency .times. .times. ( MHz
) ( Eq . .times. #10 ) ##EQU00009##
The results of the measurements are shown in FIG. 4.
[0097] FIG. 4 is a graph showing an observed relationship between
initial diameter of a droplet and the mechanical index required to
vaporize it. Droplets with diameters in the low micron range were
seen to vaporize as an approximately logarithmic function of
initial diameter. Droplets near the optical resolution limit of the
experimental setup could be vaporized with brief 2 .mu.s pulses at
mechanical indices well-below the current clinical limit of 1.9 for
diagnostic ultrasound imaging. Upon exposure to ultrasonic energy,
vaporization of the largest content present in the
samples--droplets larger than 1 .mu.m, which could be resolved
optically--was achieved at clinically relevant pressures such that
a logarithmic relationship between initial diameter and pressure
required to vaporize could be observed. As predicted, the pressure
required to vaporize droplets was inversely related to droplet
diameter.
[0098] Droplets produced by the droplet extrusion and bubble
condensation methods were measured optically before and after
activation, and it was observed that the resulting increase in
volume after vaporization was close to that predicted by ideal gas
laws (approximately 5 to 6 times the original droplet
diameter).
Tunable Activation Energy
[0099] Droplets generated according to the methods described herein
have activation energies that depend on the size of the droplet and
the substance encapsulated in the droplet. For example, droplets
containing pure DFB, which has a boiling point of -1.7.degree. C.,
has a higher activation energy than droplets containing pure OFP,
which has a boiling point of -37.6.degree. C. By creating droplets
that contain a mixture of two substances each having a different
boiling point, it is possible to create a droplet having a custom
activation energy. This is shown in FIG. 5.
[0100] FIG. 5 is a graph showing mechanical index as a function of
droplet diameter as observed using samples of droplets containing
three different substances: DFB, OFP, and a 50%/50% mix of DFB and
OFP, generated using the bubble condensation method according to an
embodiment of the subject matter described herein. Due to the lower
boiling point of OFP, a greater ambient pressure was needed before
condensation of the sample was observed. In one embodiment, the DFP
droplets produced had mean diameters of 360.+-.156 nm (N=3). Also,
due to the equipment used to verify the experimental results, only
droplets larger than 1 micron were tested, but it is expected that
sub-micron droplets would show analogous behavior. Droplets
composed of a 50/50 mixture of DFB and OFP showed ultrasonic
vaporization thresholds between that of each `pure` perfluorocarbon
at room temperature under the same test conditions.
[0101] It can be seen from the graph in FIG. 5 that droplets
containing only DFB required a mechanical index of approximately
1.3 to activate, droplets containing only OFP required a mechanical
index of approximately 0.8 to activate, and droplets containing the
50/50 mixture required a mechanical index in between 0.8 and 1.3,
in the 1.1.about.1.2 range. By adjusting the mix of a relatively
more volatile substance with a relatively less volatile substance,
e.g., OFP and DFP, a droplet may be produced that has an activation
energy that is somewhere in between the activation energies of the
individual components of the mix. Thus, the energy required to
vaporize a nanodroplet can be manipulated by simply mixing the
gases to a desired ratio prior to condensation.
[0102] At both room and body temperature, DFB and OFP droplets were
vaporized in vitro with waveforms similar to those found on
clinical diagnostic ultrasound machines, and with pressures less
than the current FDA limit at 8 MHz (approximately 5.4 MPa).
Unexpectedly, OFP showed relative stability at room temperature
(nearly 60.degree. C. above its natural boiling point), but reacted
highly upon exposure to body temperature. DFB droplets, on the
other hand, showed remarkable stability at both room and body
temperature. As expected, droplet stability correlated inversely
with boiling point.
Applications
[0103] FIG. 6 is a flow chart illustrating an exemplary process for
delivery of particles to a target region according to an embodiment
of the subject matter described herein. In the embodiment
illustrated in FIG. 6, at block 600, particles comprising stable,
activatable nanodroplets, each nanodroplet comprising a liquid
encapsulated in a shell, wherein the liquid comprises at least one
component that is a gas at room temperature and atmospheric
pressure, are introduced into a blood vessel in the vicinity of a
target region, and at 602, the method includes waiting until a
sufficient amount of particles have extravasated into the target
region. In one embodiment, the target region is exposed to some
form of activation energy, such as ultrasonic, mechanical, thermal,
or radio frequency energy, activating the nanodroplets and causing
them to expand into microbubbles.
[0104] FIG. 7 is a flow chart illustrating an exemplary process for
medical diagnostic imaging using activatable droplets as contrast
agents according to an embodiment of the subject matter described
herein. In the embodiment illustrated in FIG. 7, at block 700,
encapsulated droplets, each encapsulated droplet comprising a
liquid encapsulated in a shell, wherein the liquid comprises a
liquid phase of a fluorocarbon, a perfluorocarbon, a
chlorofluorocarbon, a hydrofluorocarbon, a hydrocarbon, a gas
having a boiling point that is below room temperature (25.degree.
C.), or combinations thereof are produced. These droplets may be
produced by using the droplet extrusion or bubble condensation
methods described above, for example. At block 702, the
encapsulated droplets so produced are introduced into a tissue to
be imaged. In one embodiment, the droplets may be introduced into a
blood vessel that is in proximity to the tissue to be imaged, which
allows the droplets to extravasate into the interstitial area of
the tissue. At block 704, activation energy sufficient to cause the
liquid within the encapsulated droplets to change from a liquid
phase to a gas phase is provided, causing the encapsulated droplets
to increase in size and become bubbles of encapsulated gas. At
block 706, ultrasonic imaging of the tissue is performed using the
bubbles as a contrast agent.
[0105] In one embodiment, a method for medical diagnostic imaging
using activatable droplets as contrast agents includes producing
encapsulated droplets, each encapsulated droplet made up of a
liquid encapsulated in a shell, where the liquid comprises a liquid
phase of a fluorocarbon, a perfluorocarbon, a chlorofluorocarbon, a
hydrofluorocarbon, a hydrocarbon, a gas having a boiling point that
is below room temperature (25.degree. C.), or combinations of the
above. The encapsulated droplets are introduced into a tissue to be
imaged, and activation energy sufficient to cause the liquid within
the encapsulated droplets to change from a liquid phase to a gas
phase is provided, causing the encapsulated droplets to increase in
size and become bubbles of encapsulated gas. Ultrasonic imaging of
the tissue using the bubbles as a contrast agent is performed. In
one embodiment, the shell comprises a lipid, protein, polymer, gel,
surfactant, peptide, or sugar. In one embodiment, the shell
comprises lung surfactant proteins or their peptide components to
form and stabilize bilayer and multilayer folds of the
encapsulation material attached to maintain enough encapsulation
material sufficient to fully encapsulate the liquid phase before
droplet vaporization and the gas phase following vaporization. In
one embodiment, providing activation energy sufficient to cause the
liquid within the encapsulated droplets to change from a liquid
phase to a gas phase includes subjecting the encapsulated droplets
to ultrasonic, X-ray, optical, infrared, microwave, or radio
frequency energy. In one embodiment, the encapsulating material
contains a chemical substance that causes the encapsulated droplets
to attach to cells of the tissue to be imaged. In one embodiment,
the tissue to be imaged comprises cancerous or pre-cancerous cells
and wherein the chemical substance attaches to proteins expressed
by the cancerous or pre-cancerous cells.
[0106] This technology is amenable to not only ultrasound imaging,
but drug and gene delivery and therapy as well. The low
concentrations of lipids (0.75-3.0 mg/mL) utilized to stabilize the
DFB droplets in one embodiment makes these formulations more
amenable to human use while also minimizing the possibility of
toxicity or bioeffects.
[0107] FIG. 8 is a flow chart illustrating an exemplary process for
medical therapy using activatable droplets as a vehicle for
delivering therapeutic agents according to an embodiment described
herein. In the embodiment illustrated in FIG. 8, at block 800,
encapsulated droplets, each encapsulated droplet comprising a
liquid encapsulated in a shell, wherein the liquid comprises a
liquid phase of a fluorocarbon, a perfluorocarbon, a
chlorofluorocarbon, a hydrofluorocarbon, a hydrocarbon, a gas
having a boiling point that is below room temperature (25.degree.
C.), or combinations thereof are produced. These droplets may be
produced by using the droplet extrusion or bubble condensation
methods described above, for example. At block 802, a therapeutic
agent is included in or on the shell. In one embodiment, the
droplets are produced first and the therapeutic agent is added into
or onto the shell afterwards. In another embodiment, the
therapeutic agent is included in the encapsulating material prior
to encapsulation, such that therapeutic agent is present within the
shell from the instant that the droplet is created. At block 804,
the encapsulated droplets are delivered to a target region. At
block 806, activation energy sufficient to cause the liquid within
the encapsulated droplets to change from a liquid phase to a gas
phase is provided, causing the encapsulated droplets to increase in
size and become bubbles of encapsulated gas, and the therapeutic
agent enters into one or more cells within the target region.
[0108] In one embodiment, a method for medical therapy using
activatable droplets includes producing encapsulated droplets, each
encapsulated droplet made up of a liquid encapsulated in a shell,
where the liquid includes a liquid phase of a fluorocarbon, a
perfluorocarbon, a chlorofluorocarbon, a hydrofluorocarbon, a
hydrocarbon, a gas having a boiling point that is below room
temperature (25.degree. C.), or a combination of the above, and
where a therapeutic agent is included in or on the shell. The
encapsulated droplets are delivered to a target region, and
activation energy sufficient to cause the liquid within the
encapsulated droplets to change from a liquid phase to a gas phase
is provided, causing the encapsulated droplets to increase in size
and become bubbles of encapsulated gas, and where the substance to
be delivered to the target tissue enters into the cells of the
target tissue. In one embodiment, the shell comprises a lipid,
protein, polymer, gel, surfactant, peptide, or sugar. In one
embodiment, the shell comprises lung surfactant proteins or their
peptide components to form and stabilize bilayer and multilayer
folds of the encapsulation material attached to maintain enough
encapsulation material sufficient to fully encapsulate the liquid
phase before droplet vaporization and the gas phase following
vaporization. In one embodiment, providing activation energy
sufficient to cause the liquid within the encapsulated droplets to
change from a liquid phase to a gas phase includes subjecting the
encapsulated droplets to ultrasonic, X-ray, optical, infrared,
microwave, or radio frequency energy. In one embodiment, the shell
contains a chemical substance that causes the encapsulated droplets
to attach to cells of the target tissue. In one embodiment, the
shell contains a net negative or net positive charge to prevent
aggregation and coalescence. In one embodiment, the shell contains
a polymeric brush layer to prevent aggregation and coalescence. In
one embodiment, the shell contains a chemical substance that causes
plasmids or genes to attach to the shell. In one embodiment, the
chemical substance is a cationic chemical. In one embodiment, the
genes are targeted for gene or plasmid delivery to a cell.
[0109] In one embodiment, the target region comprises cancerous or
pre-cancerous cells and wherein the chemical substance attaches to
proteins expressed by the cancerous or pre-cancerous cells. In one
embodiment, the encapsulated droplets are delivered to the target
region by being introduced into a blood vessel in the vicinity of
the target region and the encapsulated droplets extravasate into
the target region. In one embodiment, the substance to be delivered
to the target region enters into the cells of the target region via
sonoporation, vaporization, endocytosis, or contact-facilitated
diffusion. In one embodiment, the substance to be delivered to the
target region includes a drug to be delivered to the target region
and/or genetic material to be inserted into the cells of the target
region.
[0110] In one embodiment, tissue-specific targeting ligands may be
incorporated into the shell of nanodroplets. For example,
tissue-specific targeting ligands may be incorporated into the
shell of microbubbles produced and later condensed into
nanodroplets according to the bubble condensation methods described
above. In one embodiment, targeted DFB microbubbles were fabricated
with DSPC, PEG, and PEG conjugated with a cyclic RGD peptide, which
is known to target .alpha..sub.v.beta..sub.3, a known angiogenic
biomarker. Likewise, control microbubbles were fabricated with
DSPC, PEG and PEG conjugated with a cyclic RAD peptide, which does
not bind to .alpha..sub.v.beta..sub.3. Targeted and non-targeted
microbubbles were condensed into nanodroplets and incubated (15
minutes) independently with cover slips confluent with human
umbilical vein endothelial cells (HUVEC), which overexpress
.alpha..sub.v.beta..sub.3 integrin. After incubation, each cover
slip was washed with cell media to remove any non-targeted
droplets. Next, each cover slip was placed on a custom built holder
and submerged in a water tank full of phosphate buffered saline
heated to 37.degree. C. for acoustic vaporization and testing. A
linear array transducer was used to take 2D cross-sectional images
across the cover slip as a baseline before vaporization. Then, the
transducer was scanned at a constant speed of 2.5 mm/s across the
cover slip at a mechanical index of 1.9 in power Doppler mode to
vaporize any adherent droplets. Finally, 2D acquisitions across the
cover slip were obtained with the transducer in contrast mode to
determine the degree of contrast (via microbubbles from droplet
vaporization) for both control and targeted samples. The brightness
of adherent microbubbles was assumed to be correlated with the
degree of .alpha..sub.v.beta..sub.3 expression. Analysis of
ultrasound images shows that incorporating targeting ligands (in
this case, the cyclic RGD peptide) increased the number of droplets
present on the cell layer (HUVECs) dramatically. After being
exposed to pressures within the limit of what a clinical ultrasound
machine can provide, any droplets adhering to the cell layer were
vaporized into microbubbles, which show up brightly on the
ultrasound scan. Comparing targeted droplets to non-targeted
droplets shows that the contrast present after vaporization was
significantly greater for targeted droplets than for non-targeted
droplets, indicating that 1) the targeting ligand was successfully
preserved in the nanodroplet shell and 2) the targeted nanodroplets
preferentially adhered to the cell layer. These results suggest
successful `transformation` of targeted microbubbles into targeted
nanodroplets, which could be valuable for applications such as
early detection and diagnosis of angiogenesis.
[0111] Delivering a droplet to targeted cells or to cells in a
targeted region may involve more than simply delivering the
substance to the exterior of the cell or into the vicinity of the
cell. In one embodiment, the droplets may be coated with a material
that causes the cells to internalize the droplets, such as via
endocytosis or phagocytosis. For example, coating a droplet with
folate may cause a cell to internalize the droplet. Once inside the
cell, activation of the droplet causes the droplet to expand to
microbubble size. In one embodiment, the activated droplet kills
the cell, either by the expansion alone, or by subsequent
excitation of the microbubble within the cell. Alternatively, the
activated droplet may not kill the cell but instead deliver the
intended payload more efficiently within the cell, e.g., by
increasing the surface area of a shell containing a therapeutic
substance intended for the cell to receive and use. In this manner,
any substance that may be delivered into the presence of the cell
(e.g., a drug to be delivered to the target region, genetic
material to be inserted into the cells of the target region, and
others) may instead be delivered into the interior of the cell
directly.
[0112] In one embodiment, the droplet may be coated with a material
that targets specific parts of the cell once internalized. For
example, the droplet may be coated with a material that causes the
droplet to attach itself, be internalized by, or otherwise target a
subcellular organelle (e.g., mitochondria). In one embodiment,
activating the droplet compromises the targeted organelle and
subsequently destroying the cell, arresting the cell's growth, or
otherwise killing the cell. In one embodiment, the droplet or its
shell could contain a chemical substance which triggers cell
apoptosis. Alternatively, the activated droplet may more
efficiently deliver a chemical substance to the targeted
organelle.
[0113] FIG. 9 is a flow chart illustrating an exemplary process for
medical therapy using activatable droplets to obstruct the flow of
blood, oxygen, or nutrients to cells, such as tumor tissues,
according to an embodiment of the subject matter described herein.
In the embodiment illustrated in FIG. 9, at block 900, encapsulated
droplets, each encapsulated droplet comprising a liquid
encapsulated in a shell, wherein the liquid comprises a liquid
phase of a fluorocarbon, a perfluorocarbon, a chlorofluorocarbon, a
hydrofluorocarbon, a hydrocarbon, a gas having a boiling point that
is below room temperature (25.degree. C.), or combinations thereof
are produced. These droplets may be produced by using the droplet
extrusion or bubble condensation methods described above, for
example. At block 902, the encapsulated droplets are delivered to a
target region. At block 904, activation energy sufficient to cause
the liquid within the encapsulated droplets to change from a liquid
phase to a gas phase is provided, causing the encapsulated droplets
to increase in size and become bubbles of encapsulated gas, and the
bubbles obstruct the flow of blood, oxygen, or nutrients to cells
within the target region. In one embodiment, the droplets are small
enough to extravasate into the interstitial space of tissues in the
target region, and the activated bubbles prevent nutrients from
passing to the tissue from blood vessels. In one embodiment, the
droplets remain within the blood vessels and, when activated, are
large enough to obstruct blood flow through the blood vessels,
which also may starve tissue within the target region. Example
target regions may include tumors, cancerous cells, or
pre-cancerous cells.
[0114] FIG. 10 is a flow chart illustrating an exemplary process
for size selection of particles according to an embodiment
described herein. In the embodiment illustrated in FIG. 10, at
block 1000, droplets or emulsions having at least one component
that is a gas at room temperature and atmospheric pressure
encapsulated in liquid form inside a lipid, protein, or polymer
capsule particles are exposed to a pressure other than atmospheric
pressure and/or a temperature other than room temperature, which
causes some portion of the particle distribution to become
activated. At block 1002, the activated particles can then be
separated from the non-activated particles. This is fairly easy to
do since bubbles are more buoyant than droplets. This technique
allows the particles to be selectively separated according to
droplet size. The larger droplets have a lower activation energy
than the smaller droplets, and so the larger droplets may be
separated from the other droplets by applying an activation energy
that is high enough to activate the larger droplets but not high
enough to activate the smaller droplets. If smaller droplets are
desired, this technique can be used to cull larger droplets from
the mix. If larger droplets are desired, the technique can be used
to harvest the larger droplets by causing them to inflate into
bubbles, scooping the now floating bubbles from the top, and
subject them to cooling and/or compression to cause them to revert
to droplet form.
[0115] In one embodiment, a method of size selection of particles
comprising droplets or emulsions having at least one component that
is a gas at room temperature and atmospheric pressure encapsulated
in liquid form inside a lipid, protein, polymer, gel, surfactant,
peptide, or sugar capsule includes exposing the particles to
pressure other than atmospheric pressure thereby causing some
portion of the particle distribution to become activated, and
separating the activated particles from the non-activated
particles. In one embodiment, exposing the particles to pressure
other than atmospheric pressure includes exposing the particles to
a pressure that is 0.about.10 mm Hg below atmospheric pressure. In
one embodiment, exposing the particles to pressure other than
atmospheric pressure includes exposing the particles to a pressure
that is 10.about.50 mm Hg below atmospheric pressure. In one
embodiment, exposing the particles to pressure other than
atmospheric pressure includes exposing the particles to a pressure
that is 50.about.100 mm Hg below atmospheric pressure. In one
embodiment, exposing the particles to pressure other than
atmospheric pressure includes exposing the particles to a pressure
that is 100.about.200 mm Hg below atmospheric pressure. In one
embodiment, exposing the particles to pressure other than
atmospheric pressure includes exposing the particles to a pressure
that is 200.about.400 mm
[0116] Hg below atmospheric pressure. In one embodiment, exposing
the particles to pressure other than atmospheric pressure includes
exposing the particles to a pressure that is 400.about.600 mm Hg
below atmospheric pressure. In one embodiment, exposing the
particles to pressure other than atmospheric pressure includes
exposing the particles to a pressure that is 600.about.800 mm Hg
below atmospheric pressure.
[0117] In one embodiment, a method of size selection of particles
comprising droplets or emulsions having at least one component that
is a gas at room temperature and atmospheric pressure encapsulated
in liquid form inside a lipid, protein, polymer, gel, surfactant,
peptide, or sugar capsule includes exposing the particles to a
temperature range thereby causing some portion of the particle
distribution to become activated, and separating the activated
particles from the non-activated particles. In one embodiment,
exposing the particles to a temperature range includes exposing the
particles to a temperature that is 0.about.60 degrees C. above room
temperature. In one embodiment, exposing the particles to a
temperature range includes exposing the particles to a temperature
that is 10.about.60 degrees C. above room temperature. In one
embodiment, exposing the particles to a temperature range includes
exposing the particles to a temperature that is 20.about.80 degrees
C. above room temperature.
[0118] In one embodiment, a method of generating microbubbles in a
biological media using a metastable nanoparticle containing a
stabilized fluorocarbon with a boiling point below the temperature
of the biological media and activating the nanoparticle, causing
the nanoparticle to transform into a microbubble. In one
embodiment, the boiling point is between 0 and 60 degrees C. below
the temperature of the biological media. In one embodiment, the
boiling point is between 10 and 60 degrees C. below the temperature
of the biological media. In one embodiment, the boiling point is
between 20 and 80 degrees C. below the temperature of the
biological media. In one embodiment, the boiling point is between
30 and 80 degrees C. below the temperature of the biological media.
In one embodiment, the boiling point is between 40 and 60 degrees
C. below the temperature of the biological media. In one
embodiment, the boiling point is between 50 and 60 degrees C. below
the temperature of the biological media.
[0119] In one embodiment, a method for medical therapy using
activatable droplets includes producing encapsulated droplets, each
encapsulated droplet made up of a liquid encapsulated in a shell,
where the liquid includes a liquid phase of a fluorocarbon, a
perfluorocarbon, a chlorofluorocarbon, a hydrofluorocarbon, a
hydrocarbon, a gas having a boiling point that is below room
temperature (25.degree. C.), or combinations of the above. The
encapsulated droplets are delivered to a target tissue, and
activation energy sufficient to cause the liquid within the
encapsulated droplets to change from a liquid phase to a gas phase
is provided, causing the encapsulated droplets to increase in size
and become bubbles of encapsulated gas. The bubbles so generated
obstruct the flow of blood, oxygen, or nutrients to a target
region. In one embodiment, the shell includes a lipid, protein,
polymer, gel, surfactant, peptide, or sugar. In one embodiment, the
shell includes lung surfactant proteins or their peptide components
to form and stabilize bilayer and multilayer folds of the
encapsulation material attached to maintain enough encapsulation
material sufficient to fully encapsulate the liquid phase before
droplet vaporization and the gas phase following vaporization. In
one embodiment, providing activation energy sufficient to cause the
liquid within the encapsulated droplets to change from a liquid
phase to a gas phase includes subjecting the encapsulated droplets
to ultrasonic, X-ray, optical, infrared, microwave, or radio
frequency energy. In one embodiment, the target region includes a
tumor, cancerous cells, or pre-cancerous cells. In one embodiment,
the encapsulating material contains a chemical substance that
causes the encapsulated droplets to attach to cells of a tissue
within the cells of the target region. In one embodiment, the
encapsulating material contains a chemical substance that causes
the encapsulated droplets to attach to cells and promote
intracellular uptake. In one embodiment, the chemical substance
attaches to proteins expressed by cells within the target
region.
CONCLUSION
[0120] It has been shown that highly volatile PFCs, such as DFB and
OFP, can be successfully generated as lipid-encapsulated micron and
sub-micron sized droplets that remain stable at physiological
temperatures. Most studies of phase-change contrast agents to date
have chosen PFCs that are stable at room temperature, presumably
due to simplicity of droplet generation. This study is the first,
to the knowledge of the authors, which has explored the use of
lower boiling-point PFCs by means of using shell encapsulation to
produce stable liquid droplets of PFCs which are normally gas at
room and body temperature. DFB-based phase-change contrast agents
show significant potential for applications such as intra-tumoral
deposition of chemotherapeutics and the imaging of interstitial
space.
[0121] It has also been shown that pressurization and
temperature-induced condensation of pre-formed microbubbles is both
an effective and advantageous means of producing contrast agents
for ADV applications compared to conventional extrusion and
emulsion-based methods for some PFCs. The samples formed at a lipid
concentration of 3 mg/mL produced a high number of viable
nanodroplets that could be vaporized at clinically feasible
pressures, resulting in a distribution of contrast-providing
microbubbles well-correlated to the original microbubble sample.
This method also may have advantages with regard to
commercialization of ADV technology, as nanodroplets can be formed
easily by adding a simple technique after traditional microbubble
preparation. Results have demonstrated that ADV of submicron sized
droplets can be induced in vitro with pressures available to
clinical diagnostic ultrasound machines.
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